Mechanobiology studies the effect of physical forces on biological tissue. Directly correlating mechanical and structural information, however, presents a major challenge for both existing imaging and for technologies that provide for characterizing the response of tissue to mechanical force. In the context of mechanobiology, optical microscopy techniques provide noninvasive imaging of biological specimens such as cells, and the extracellular matrix. However, optical imaging methods cannot provide information about the mechanical properties of the imaged tissue. The closest approach to the use of optical techniques to obtain mechanical data has come from the use of optical tweezers (a non-imaging technique) as a force probe. However, that approach is limited to maximum applied forces on the order of pN and are not appropriate for tissue-level testing—thereby limiting the technique to a very narrow window of problems in biology.
The various modalities for characterizing measures of mechanical properties for soft tissues can be made using several modalities, but each has its drawbacks. Modalities for characterizing measures of mechanical properties include bulk testing (e.g. tension, compression), macroscale indentation (i.e. hardness testing), atomic force microscopy (AFM), and instrumented nanoindentation (NI). Drawbacks associated with bulk testing include the requirements of sample preparation appropriate to the testing mode and the inability to measure local properties. For example, for tensile tests one needs to grip the sample at both ends. This is difficult for most tissues as they are soft and slippery, and therefore can slide out of the grips if not gripped tightly enough, or, tissue may be damaged if gripped too intensely. Furthermore, all tissues are inherently heterogeneous in structure (and, therefore, properties), often over only 100s of microns. This heterogeneity is lost in bulk testing since the data represent an average over the entire sample, which is more typically in the size range of millimeters or centimeters. Local measures, such as AFM and NI, however, provide for capturing this heterogeneity.
AFM was developed primarily for high-resolution (nm scale) topographical surface profiling. Quantification of mechanical properties using AFM is complicated because probe stiffness and geometry vary between probes, and thus each must be accurately characterized in order to have accurate force measurements. Specifically, the cantilever stiffness and the tip geometry must each be measured. Since cantilevers often last only one test sample, cantilever stiffness must be calibrated with each sample.
Nanoindentation has been used to characterize mechanical properties of a sample, such as the Young's modulus as a function of depth, etc., since the early 1990s. NI has also been accompanied by scanning probe imaging techniques in order to elucidate morphological characteristics of the surface undergoing NI. However, it has not been possible, heretofore, to apply optical techniques, whether for imaging in scatter, or for non-linear optical modalities such as second-harmonic generation (SHG), for example, to the identical region of a sample subject to NI. This has proven to be a severe limitation of the NI technique.
It would be highly desirable, therefore, to provide researchers, especially in the study of biological tissue, with an instrument that enables concurrent NI and optical access to the same region of a probed sample. A device that provides such functionality is described for the first time herein.